OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxy...OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.展开更多
OBJECTIVE To explore the effects and mechanism of icariside Ⅱ(ICS Ⅱ),a pharmacologically active compound derived from herbal Epimedii with previous study-proved phosphodiesterase 5(PDE5) inhibitors,was investigated ...OBJECTIVE To explore the effects and mechanism of icariside Ⅱ(ICS Ⅱ),a pharmacologically active compound derived from herbal Epimedii with previous study-proved phosphodiesterase 5(PDE5) inhibitors,was investigated in vivo using a middle cerebral artery occlusion/reperfusion(MCAO/R) model in rats and in vitro using an oxygen-glucose deprivation/reperfusion(OGD/R) model in primary hippocampal neurons.METHODS Laser Doppler flowmeter was introduced to examine the cerebral blood flow of MCAO/R rats.The neurological deficits scores,brain water content and infarction volume were assessed after MCAO/R.OGD/R-induced primary hippocampal neuronal injury and apoptosis were examined by MTT,lactate dehydrogenase(LDH) release,TUNEL staining and flow cytometry,respectively.Expressions of PDE5 A and memory-related signaling pathways were measured using Western blotting analysis.The direct interaction between ICS Ⅱand PDE5 was further evaluated by molecular docking.RESULTS ICS Ⅱ significantly decreased the infraction volume in MCAO/R rats.Furthermore,ICS Ⅱ significantly abrogated OGD/R-induced hippocampal neuronal death.Moreover,ICSⅡ not only effectively restored the 3′ 5′-cyclic guanosine monophosphate(cGMP) level and protein kinase G(PKG) activity both in vivo and in vitro,but also increased brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrkB) and cAMP response element-binding protein(CREB) expressions,thereby inhibited hippocampal neuronal apoptosis.Mechanistically,the beneficial effects of ICS Ⅱ was attributed to its activation of the PKG/TrkB/BDNF via increasing BDNF expression,evidenced by that the inhibition effects of ICSⅡ was abrogated by Rp-8-BrcGMPS,a PKG inhibitor,or ANA-12,a TrkB inhibitor.ICSⅡ also decreased both protein level and activity of PDE5.Notably,ICSⅡ might effectively bind and inhibite PDE5 as demonstrated by relatively high binding score.CONCLUSION ICSⅡ significantly protect against cerebral ischemia/reperfusion injury in rats and rescues OGD/Rinduced hippocampal neuronal injury,and the underling mechanisms are,at least partly,due to inhibition of PDE5 and activation of BDNF/TrkB/CREB signaling pathway.Hence ICS Ⅱ may be an effective agent for combating cerebral ischemia/reperfusion injury.展开更多
目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、...目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、高剂量组(n=3),除对照组外,其余4组建立OGD/R神经元细胞损伤模型,低、中、高剂量组分别给予阿司匹林100、200、400μg/ml处理。检测各组细胞活力及炎性因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素(interleukin,IL)1β、IL-6水平;试剂盒检测超氧化物歧化酶、过氧化氢酶、谷胱甘肽、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛水平;Western blot检测铁死亡相关蛋白溶质载体家族7成员11(solute carrier family 7 members 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)以及酰基辅酶A合成酶长链家族成员4(acyl-coa synthase long chain family member 4,ACSL4)水平。结果模型组细胞活力明显低于对照组,差异有统计学意义(0.49±0.07 vs 1.00±0.12,P<0.01),低、中、高剂量组细胞活力明显高于模型组,差异有统计学意义(0.72±0.10 vs 0.49±0.07,P<0.05;0.87±0.10 vs 0.49±0.07,P<0.01;0.93±0.07 vs 0.49±0.07,P<0.01)。与对照组比较,模型组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛、ACSL4蛋白表达明显升高,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显降低,差异有统计学意义(P<0.01)。与模型组比较,低、中、高剂量组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶Fe^(2+)、丙二醛、ACSL4蛋白表达明显降低,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显升高,差异有统计学意义(P<0.05,P<0.01)。结论阿司匹林可以通过调节铁死亡,减轻OGD/R诱导的小鼠神经元HT22细胞损伤,且呈剂量依赖性。展开更多
基金National Natural Science Foundation of China(81560666)Program for Excellent Young Talents of Zunyi Medical Uiverstity(15zy-002)+1 种基金Science and Technology Innovation Talent Team of Guizhou Province(20154023)the ″Hundred″Level of High-level Innovative Talents in Guizhou Province(QKHRCPT 20165684)；and Program forChangjiang Scholars and Innovative ResearchTeam in University of China(IRT一17R113).
文摘OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.
基金National Natural Science Foundation of China(81560585)Program for Excellent Young Talentsof Zunyi Medical University(15zy-002)+2 种基金Scienceand Technology Innovation Talent Team of GuizhouProvince(20154023)the hundred”Level of High—level Innovative Talents in Guizhou Province(QKHRCPT 20165684):Education Department of Guizhou Province of China[GNYL(2017)006,YLXKJS—YS一06]Program for Changjiang Scholars and lnnovative Research Team in University,China(IRT-17R113).
文摘OBJECTIVE To explore the effects and mechanism of icariside Ⅱ(ICS Ⅱ),a pharmacologically active compound derived from herbal Epimedii with previous study-proved phosphodiesterase 5(PDE5) inhibitors,was investigated in vivo using a middle cerebral artery occlusion/reperfusion(MCAO/R) model in rats and in vitro using an oxygen-glucose deprivation/reperfusion(OGD/R) model in primary hippocampal neurons.METHODS Laser Doppler flowmeter was introduced to examine the cerebral blood flow of MCAO/R rats.The neurological deficits scores,brain water content and infarction volume were assessed after MCAO/R.OGD/R-induced primary hippocampal neuronal injury and apoptosis were examined by MTT,lactate dehydrogenase(LDH) release,TUNEL staining and flow cytometry,respectively.Expressions of PDE5 A and memory-related signaling pathways were measured using Western blotting analysis.The direct interaction between ICS Ⅱand PDE5 was further evaluated by molecular docking.RESULTS ICS Ⅱ significantly decreased the infraction volume in MCAO/R rats.Furthermore,ICS Ⅱ significantly abrogated OGD/R-induced hippocampal neuronal death.Moreover,ICSⅡ not only effectively restored the 3′ 5′-cyclic guanosine monophosphate(cGMP) level and protein kinase G(PKG) activity both in vivo and in vitro,but also increased brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrkB) and cAMP response element-binding protein(CREB) expressions,thereby inhibited hippocampal neuronal apoptosis.Mechanistically,the beneficial effects of ICS Ⅱ was attributed to its activation of the PKG/TrkB/BDNF via increasing BDNF expression,evidenced by that the inhibition effects of ICSⅡ was abrogated by Rp-8-BrcGMPS,a PKG inhibitor,or ANA-12,a TrkB inhibitor.ICSⅡ also decreased both protein level and activity of PDE5.Notably,ICSⅡ might effectively bind and inhibite PDE5 as demonstrated by relatively high binding score.CONCLUSION ICSⅡ significantly protect against cerebral ischemia/reperfusion injury in rats and rescues OGD/Rinduced hippocampal neuronal injury,and the underling mechanisms are,at least partly,due to inhibition of PDE5 and activation of BDNF/TrkB/CREB signaling pathway.Hence ICS Ⅱ may be an effective agent for combating cerebral ischemia/reperfusion injury.
文摘目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、高剂量组(n=3),除对照组外,其余4组建立OGD/R神经元细胞损伤模型,低、中、高剂量组分别给予阿司匹林100、200、400μg/ml处理。检测各组细胞活力及炎性因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素(interleukin,IL)1β、IL-6水平;试剂盒检测超氧化物歧化酶、过氧化氢酶、谷胱甘肽、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛水平;Western blot检测铁死亡相关蛋白溶质载体家族7成员11(solute carrier family 7 members 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)以及酰基辅酶A合成酶长链家族成员4(acyl-coa synthase long chain family member 4,ACSL4)水平。结果模型组细胞活力明显低于对照组,差异有统计学意义(0.49±0.07 vs 1.00±0.12,P<0.01),低、中、高剂量组细胞活力明显高于模型组,差异有统计学意义(0.72±0.10 vs 0.49±0.07,P<0.05;0.87±0.10 vs 0.49±0.07,P<0.01;0.93±0.07 vs 0.49±0.07,P<0.01)。与对照组比较,模型组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛、ACSL4蛋白表达明显升高,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显降低,差异有统计学意义(P<0.01)。与模型组比较,低、中、高剂量组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶Fe^(2+)、丙二醛、ACSL4蛋白表达明显降低,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显升高,差异有统计学意义(P<0.05,P<0.01)。结论阿司匹林可以通过调节铁死亡,减轻OGD/R诱导的小鼠神经元HT22细胞损伤,且呈剂量依赖性。
